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Biological Systems for Hydrogen Photoproduction

Maria L. Ghirardi, PI National Renewable Energy Laboratory

May 16, 2013 PD037 This presentation does not contain any proprietary, confidential, or otherwise restricted information

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Overview

Timeline • Project start date: FY00 • Project end date: 9/30/2013* • Percent complete: 85% *Project continuation and direction determined annually by DOE

Budget

• Total project funding: $10,551K • Funding received in FY12: $600K • Planned funding for FY13: $480K

Barriers • Barriers addressed:

- Rate of H2 production (AO) - Oxygen Accumulation (AP) • Targets: - Duration of production

- Solar to H2 (STH) energy conversion

Partners (in FY12) • Dr. Sergey Kosourov, Institute of Basic Biological Problems, RAS, Pushchino, Russia • Dr. Eric Johnson, Johns Hopkins University

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Relevance Photobiological water splitting coupled to hydrogenase-mediated H2 production has the potential to convert about 10% of incident solar energy into H2. Various barriers have been identified as currently limiting green algal H2 production, including: • the O2 sensitivity of the hydrogenase enzyme, • the competition for reductant with CO2 fixation and cyclic electron flow, • the down-regulation of photosynthesis due to non-dissipation of the proton gradient and state transitions, and • the low light-saturation of photosynthesis.

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Objectives • General goal: Develop photobiological systems for large-scale, low cost

and efficient H2 production from water (barriers AO and AP).

• Specific tasks: Task 1: Address the O2 sensitivity of hydrogenases that prevent continuity

of H2 photoproduction under aerobic, high solar-to-hydrogen (STH) conversion efficiency conditions.

Task 2: Utilize a limited STH H2-producing method (sulfur deprivation) as a platform to address or test other factors limiting commercial algal H2 photoproduction, including low rates due to biochemical and engineering mechanisms – discontinued in FY13 due to budget restrictions

Technical Targets: Photolytic Biological Hydrogen Production Characteristics Units 2012

Status 2015

Target 2020

Target Ultimate

Target Duration of continuous H2 production at full sunlight intensity

Time units

N/A 30 min 4 h 8 h

Solar to H2 (STH) energy conversion ratio

% N/A 2 5 17

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Approach/Milestones – Task 1 Task 1: Address the O2 sensitivity of hydrogenase by introducing the gene encoding for a

hydrogenase from Clostridium acetobutylicum that is more O2-tolerant in vitro into the photosynthetic alga Chlamydomonas reinhardtii; measure its linkage to water oxidation and in vivo O2 tolerance.

Dr. Paul King, NREL Seth Noone, NREL

Task 1 FY12 Milestone Due date Status 3.3.5 Demonstrate expression of an active Ca1 in a C. reinhardtii

hydrogenase-less background and characterize O2-sensitivity of light-driven H2 production (PsaD-Ca1 construct)

9/30/12

Completed

FY13 Milestones Due date Status 3.3.1-1 Re-test the H2 photoproduction activity of the PsaD-Ca1

transformant strain 55 by the Clark electrode 12/12 Completed

3.3.1-2 Screen 300 HYDA-Ca1transformants for the presence of the Ca1 gene and test positive transformants for H2 production activity through the plate assay and Clark electrode

2/13

Completed

3.3.1-3 Generate new transformants with 3 variations of added introns from, respectively, RbcS2, HYDA1 and HYDA2

4/13 On track

3.3.1-4 Test at least 100 strains from the first generation of intron-containing transformants for H2-production activity through the plate assay (contingent upon finding a new post-doc to perform the work)

5/13

On track

3.3.1-5 Go/NoGo: if addition of introns increases the H2 photoproduction activity and stability of Ca1 by at least 3 times compared to HYDA1 of PsaD-based constructs, use the strain for further improvements; if not, propose a new plan for DOE’s approval to re-direct the work (CPS Agreement Milestone)

7/13

On track

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Figure 1. PCR of genomic DNA from various Ca1transformants

Ca1 transformants

Ca1

Accomplishments and Progress – Task 1 Task 1 – Expression of the more O2-tolerant clostridial Ca1 hydrogenase in C.

reinhardtii Last year’s Progress: A double hydrogenase knock-out mutant (–hydA1/hydA2) was

isolated under BES funding and served as a host for expression of CaI hydrogenase gene behind the PsaD promoter (which requires light for optimal anaerobic induction).

PCR (genomic DNA) RT-PCR (mRNA) seconds

Rela

tive

units

Light-dependent H2 production was

detected! Mutant, light induction Mutant, dark induction Knock-out strain, light induction

The clostridial hydrogenase was inserted into the Chlamydomonas

genome and transcribed.

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Accomplishments and Progress - Task 1 Task 1 – Expression of the more O2-tolerant clostridial Ca1 hydrogenase in C.

reinhardtii and measurement of its O2 tolerance - completed FY12 milestone 3.3.5.

Strain Rate of H2 production in Clark electrode (µmol H2

mg Chl-1 h-1)

% WT

WT 260 100 Double knock-out 0.09 0.035 Double knock-out/Ca1

5.1 2

Strain 55 expressing Ca1 photoproduces H2 at around 2% of WT rate

Strain Cycle of illumination

Rate of H2 production in Clark electrode

(µmol H2 mg Chl-1 h-1)

Change in rate from 1st light

cycle WT 1 269 N/A

2 205 -23% 3 179 -33%

Double knock-out/Ca1

1 5.1 N/A 2 5.6 +10% 3 6.2 +25%

Ca1 activity is not lost upon subsequent cycles of illumination, suggesting higher

tolerance to photosynthetically-produced O2

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Accomplishments and Progress - Task 1

PsaD-Ca1 Strain 55

However, GFP plate screen shows considerable heterogeneity of H2 production

levels among cells grown from a liquid culture of the Ca1-expressing transformant ...

Nov. 2011

Jan 2012

Light ON

Rela

tive

rate

0 50 100 150 200 250 300 350 400 450 500

seconds

… and activity is not stable over time if culture is serially propagated.

Task 1 – Expression of the more O2-tolerant clostridial Ca1 hydrogenase in C. reinhardtii and measurement of its O2 tolerance

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The strain shown as having the highest H2 production in the previous slide, strain 55 was re-plated, retested by GFP, and showed more uniform H2 production as shown above.

Accomplishments and Progress – Task 1 Task 1. Recover original high H2-producing strain from plates and re-test it – completion of milestone 3.3.1-1

Double knock-out WT

Double Knock Out

Ca1

!0"

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Accomplishments and Progress – Task 1 Clark H2-Production Rate versus μM O2s were plotted, and the results fitted to a bi-exponential-decay curve, according to the equation below. The two inactivation rate constants, τ1 and τ2, were estimated for WT and Ca1 cells.

Enzyme/ Strain

O2 Inactivation Rate Constants (τ1 and τ2) (μM O2-1s-1) Fast Slow

O2 Tolerance (Ratio to WT)

Fast Slow WT 2.7 x 10-2 ± 2 x 10-3 1.7 x 10-3 ± 5.2 x 10-4 NA NA

PsaDP-Ca1 1.4 x 10-3 1 x 10-4 ± 2 x 10-5 19 17

HydAP-Ca1 3 x 10-3 3.5 x 10-4 9 5

Conclusions: • In vivo expression of Ca1 in Chlamydomonas shows higher

O2 tolerance (5-19 fold) for photo-H2 production than wild-type;

• Level of increased O2 tolerance of Ca1 compared to HYDA1 in vivo is similar to differences of the purified enzymes measured in vitro.

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Strain 28

Task 1. Screen 300 HYDA-Ca1transformants for the presence of the Ca1 gene and test positive transformants for H2 production activity through the plate assay and Clark electrode – completion of milestone 3.3.1-2

Accomplishments and Progress - Task 1

Transformants 1-6

7-12 13-18 19-24 25-30 31-36

D66wt

* * * *

Strain 28 showed heterogeneity (left) in GFP H2 plate assay, and photoproduced H2 at lower rates (right) compared to strains 55

High-throughput PCR screen identified additional transformants carrying the Ca1 gene out of a library of 300

Plating and GFP screening showed two H2 producing transformants (17, 28)

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28

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Proposed Future Work

2013 Task 1 – Generate additional Ca1 expression constructs that contain Rubisco

Small Subunit or HydA1 introns and test stability and levels of Ca1 activity by GFP plate assay and Clark Electrode; complete Go/NoGo milestone demonstrating at least 3X higher activity and stability of recombinant Ca1 expressed in the C. reinhardtii double knock-out background and characterize O2 sensitivity of light-driven H2 production.

Work beyond FY13: Start to genetically combine selected traits into a strain expressing the Ca1 hydrogenase. Develop photobioreactor systems for cyclic or continuous H2 production based on current optimization of gas space composition (shown last year) and alginate immobilization.

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Task 2: Utilize the limited STH sulfur-deprivation method to test (a) the rates of H2 production by inducible ATP synthase mutants that are not limited by the non-dissipation of a proton gradient; and (b) the long-term performance of immobilized algal cultures. Discontinued in FY13

Approach/Milestones – Task 2

Task 2 FY12 Milestones Due date

Status

3.3.3 Demonstrate continuous operation of the sulfur-deprivation process for a total of 2 months upon addition of phosphate/sulfate to alginate-immobilized cultures.

9/30/12 Completed

Photo-synthesis(light)

Anaerobic glycolysis

Aerobic respiration

sugar

O2

Sulfur replete

Anaerobic glycolysis

respiration

sugar

CO2

Sulfur depleted

Formate, acetate

CO2 + H2O

Photo-synthesis

(light)

H2

Aerobic O2

H2O

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Accomplishments and Progress – Task 2 Task 2 – Demonstrate continuous H2 production for 2 months (1440 hours) by

sulfur-deprived, alginate-immobilized algae Last year’s results: alginate-immobilized algae photoproduce H2 at higher specific rates

and light conversion efficiencies than cultures in suspension upon sulfur deprivation, and they show higher tolerance to aerobic environments; cycles of +S/-S resulted in prolongation of H2 production for an additional 6 cycles of about 500 hours each.

Prolonged H2 photoproduction was demonstrated by cycles of +S/-S

+S -S

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Accomplishments and Progress – Task 2

Task 2: Demonstrate continuous operation of the sulfur deprivation process for a total of 2 months (1440 hours) upon addition of phosphate/sulfate using alginate-immobilized cultures – completed FY12 milestone 3.3.3. by 9/30/12.

Conclusions: • H2 production was observed for up to 3.75 months using the wild-type CC124 strain • The rates of production declined to very low levels after about 10 days • A different strain of Chlamydomonas, EJ12F3 showed higher rates of H2 photoproduction during the first 50 days. • The economics of the process depend on the trade-off between longer H2 production vs. lower rates

Time, hours

H 2 p

hoto

prod

uctio

n ra

te,

mm

ol m

-2 h

-1

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Collaborations

Partners (subcontractors) in FY12:

- Dr. Sergey Kosourov, Russian Academy of Sciences – applies sulfur deprivation to sulfur-immobilized C. reinhardtii cultures and tests their H2-production capabilities (Task 2).

- Dr. Eric Johnson, Johns Hopkins University – generates ATP synthase

mutants, develops transformation protocols and transforms Chlamydomonas reinhardtii; tests physiological properties of transformants (Task 2).

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Relevance: Photobiological systems required only water, CO2 and minerals for cultivation; if optimized to collect additional wavelengths of light, they will have the potential of converting 17% of the solar energy into H2 at a cost competitive with gasoline. Approach: NREL is expressing a more O2-tolerant bacterial hydrogenase in green algae and testing its in vivo tolerance to O2. Technical Accomplishments and Progress: 1. Successfully expressed a more O2-tolerant, bacterial hydrogenase in a

Chlamydomonas strain lacking native hydrogenase activity and detected more O2-tolerant photoproduction of H2 in vivo.

Collaborations: none in FY13. Proposed Future Research: Increase the recombinant hydrogenase activity and

stability in Chlamydomonas; start combining different traits to generate a more efficient H2-producing strain of Chlamydomonas.

Summary Slide

Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Accomplishments and Progress – Task 1Slide Number 7Slide Number 8Slide Number 9Slide Number 10Accomplishments and Progress – Task 1Slide Number 12Proposed Future WorkApproach/Milestones – Task 2Accomplishments and Progress – Task 2Accomplishments and Progress – Task 2CollaborationsSlide Number 18Reviewer-only SlidesResponse to Reviewers’ CommentsResponse to Reviewers’ commentsCritical Assumptions and IssuesPublicationsPresentations